1. |
- Aquila, Andrew, et al.
(author)
-
Time-resolved protein nanocrystallography using an X-ray free-electron laser
- 2012
-
In: Optics Express. - 1094-4087. ; 20:3, s. 2706-2716
-
Journal article (peer-reviewed)abstract
- We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.
|
|
2. |
- Borg, Jörgen, et al.
(author)
-
Perilipin is present in islets of Langerhans and protects against lipotoxicity when overexpressed in the beta-cell line INS-1.
- 2009
-
In: Endocrinology. - : The Endocrine Society. - 0013-7227 .- 1945-7170. ; 150:7, s. 3049-3057
-
Journal article (peer-reviewed)abstract
- Lipids have been shown to play a dual role in pancreatic beta-cells - a lipid-derived signal appears to be necessary for glucose-stimulated insulin secretion, whereas lipid accumulation causes impaired insulin secretion and apoptosis. The ability of the protein perilipin to regulate lipolysis prompted an investigation of the presence of perilipin in the islets of Langerhans. In this study evidence is presented for perilipin expression in rat, mouse and human islets of Langerhans as well as in the rat clonal beta-cell line INS-1. In rat and mouse islets, perilipin was verified to be present in beta-cells. In order to examine if the development of lipotoxicity could be prevented by manipulating the conditions for lipid storage in the beta-cell, INS-1 cells with adenoviral-mediated overexpression of perilipin were exposed to lipotoxic conditions for 72 hours. In cells exposed to palmitate, perilipin overexpression caused increased accumulation of triacylglycerols and decreased lipolysis compared to control cells. Whereas glucose-stimulated insulin secretion was retained following palmitate exposure in cells overexpressing perilipin, it was completely abolished in control beta-cells. Thus, overexpression of perilipin appears to confer protection against the development of beta-cell dysfunction following prolonged exposure to palmitate by promoting lipid storage and limiting lipolysis.
|
|
3. |
- Chapman, Henry N, et al.
(author)
-
Femtosecond X-ray protein nanocrystallography.
- 2011
-
In: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 470:7332, s. 73-7
-
Journal article (peer-reviewed)abstract
- X-ray crystallography provides the vast majority of macromolecular structures, but the success of the method relies on growing crystals of sufficient size. In conventional measurements, the necessary increase in X-ray dose to record data from crystals that are too small leads to extensive damage before a diffraction signal can be recorded. It is particularly challenging to obtain large, well-diffracting crystals of membrane proteins, for which fewer than 300 unique structures have been determined despite their importance in all living cells. Here we present a method for structure determination where single-crystal X-ray diffraction 'snapshots' are collected from a fully hydrated stream of nanocrystals using femtosecond pulses from a hard-X-ray free-electron laser, the Linac Coherent Light Source. We prove this concept with nanocrystals of photosystem I, one of the largest membrane protein complexes. More than 3,000,000 diffraction patterns were collected in this study, and a three-dimensional data set was assembled from individual photosystem I nanocrystals (∼200nm to 2μm in size). We mitigate the problem of radiation damage in crystallography by using pulses briefer than the timescale of most damage processes. This offers a new approach to structure determination of macromolecules that do not yield crystals of sufficient size for studies using conventional radiation sources or are particularly sensitive to radiation damage.
|
|
4. |
- Ekeberg, Tomas, et al.
(author)
-
Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser
- 2016
-
In: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3
-
Journal article (peer-reviewed)abstract
- Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms.
|
|
5. |
|
|
6. |
- Ekeberg, Tomas, 1983-, et al.
(author)
-
Three-dimensional structure determination with an X-ray laser
-
Other publication (other academic/artistic)abstract
- Three-dimensional structure determination of a non-crystalline virus has been achieved from a set of randomly oriented continuous diffraction patterns captured with an X-ray laser. Intense, ultra-short X-ray pulses intercepted a beam of single mimivirus particles, producing single particle X-ray diffraction patterns that are assembled into a three-dimensional amplitude distribution based on statistical consistency. Phases are directly retrieved from the assembled Fourier distribution to synthesize a three-dimensional image. The resulting electron density reveals a pseudo-icosahedral asymmetric virion structure with a compartmentalized interior, within which the DNA genome occupies only about a fifth of the volume enclosed by the capsid. Additional electron microscopy data indicate the genome has a chromatin-like fiber structure that has not previously been observed in a virus.
|
|
7. |
- Gorkhover, Tais, et al.
(author)
-
Femtosecond and nanometre visualization of structural dynamics in superheated nanoparticles
- 2016
-
In: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 10:2, s. 93-97
-
Journal article (peer-reviewed)abstract
- The ability to observe ultrafast structural changes in nanoscopic samples is essential for understanding non-equilibrium phenomena such as chemical reactions, matter under extreme conditions, ultrafast phase transitions and intense light-matter interactions. Established imaging techniques are limited either in time or spatial resolution and typically require samples to be deposited on a substrate, which interferes with the dynamics. Here, we show that coherent X-ray diffraction images from isolated single samples can be used to visualize femtosecond electron density dynamics. We recorded X-ray snapshot images from a nanoplasma expansion, a prototypical non-equilibrium phenomenon. Single Xe clusters are superheated using an intense optical laser pulse and the structural evolution of the sample is imaged with a single X-ray pulse. We resolved ultrafast surface softening on the nanometre scale at the plasma/vacuum interface within 100 fs of the heating pulse. Our study is the first time-resolved visualization of irreversible femtosecond processes in free, individual nanometre-sized samples.
|
|
8. |
- Hantke, Max F., et al.
(author)
-
A data set from flash X-ray imaging of carboxysomes
- 2016
-
In: Scientific Data. - : Springer Science and Business Media LLC. - 2052-4463. ; 3
-
Journal article (peer-reviewed)abstract
- Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth’s carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere.
|
|
9. |
- Hantke, Max F., et al.
(author)
-
High-throughput imaging of heterogeneous cell organelles with an X-ray laser
- 2014
-
In: Nature Photonics. - : Springer Science and Business Media LLC. - 1749-4885 .- 1749-4893. ; 8:12, s. 943-949
-
Journal article (peer-reviewed)abstract
- We overcome two of the most daunting challenges in single-particle diffractive imaging: collecting many high-quality diffraction patterns on a small amount of sample and separating components from mixed samples. We demonstrate this on carboxysomes, which are polyhedral cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min with the Linac Coherent Light Source running at 120 Hz. We separate different structures directly from the diffraction data and show that the size distribution is preserved during sample delivery. We automate phase retrieval and avoid reconstruction artefacts caused by missing modes. We attain the highest-resolution reconstructions on the smallest single biological objects imaged with an X-ray laser to date. These advances lay the foundations for accurate, high-throughput structure determination by flash-diffractive imaging and offer a means to study structure and structural heterogeneity in biology and elsewhere.
|
|
10. |
- Johansson, Linda C, 1983, et al.
(author)
-
Lipidic phase membrane protein serial femtosecond crystallography.
- 2012
-
In: Nature methods. - : Springer Science and Business Media LLC. - 1548-7105 .- 1548-7091. ; 9:3, s. 263-265
-
Journal article (peer-reviewed)abstract
- X-ray free electron laser (X-FEL)-based serial femtosecond crystallography is an emerging method with potential to rapidly advance the challenging field of membrane protein structural biology. Here we recorded interpretable diffraction data from micrometer-sized lipidic sponge phase crystals of the Blastochloris viridis photosynthetic reaction center delivered into an X-FEL beam using a sponge phase micro-jet.
|
|